A Caveolin Dominant Negative Mutant Associates with Lipid Bodies and Induces Intracellular Cholesterol Imbalance

Abstract

Recent studies have indicated a role for caveolin in regulating cholesterol-dependent signaling events. In the present study we have analyzed the role of caveolins in intracellular cholesterol cycling using a dominant negative caveolin mutant. The mutant caveolin protein, cav-3DGV, specifically associates with the membrane surrounding large lipid droplets. These structures contain neutral lipids, and are accessed by caveolin 1–3 upon overexpression. Fluorescence, electron, and video microscopy observations are consistent with formation of the membrane-enclosed lipid rich structures by maturation of subdomains of the ER. The caveolin mutant causes the intracellular accumulation of free cholesterol (FC) in late endosomes, a decrease in surface cholesterol and a decrease in cholesterol efflux and synthesis. The amphiphile U18666A acts synergistically with cavDGV to increase intracellular accumulation of FC. Incubation of cells with oleic acid induces a significant accumulation of full-length caveolins in the enlarged lipid droplets. We conclude that caveolin can associate with the membrane surrounding lipid droplets and is a key component involved in intracellular cholesterol balance and lipid transport in fibroblasts.

PrPc and reggies/flotillins are contained in and released via lipid-rich vesicles in Jurkat T cells

“…The more homogeneous labeling we find in peripherally located vesicles is considered realistic, because neither permeabilization nor dehydration solvents were used for cryo-EM. Internal staining of lipid droplets was also reported for caveolin in cryo-EM [20,21]. Staining at the lipid vesicle margin in immunofluorescence may result from insufficient antibody penetration which would require thorough permeabilization to allow hydrophilic antibodies to reach the vesicle interior.…”

“…Reports on caveolin-1 and cholesterol transport in FRT cells and hepatocytes have shown the association of mutant caveolin-1 with Nile Red-labeled 'lipid droplets' [15,16]. Lipid droplets, however, are thought to be surrounded by a phospholipid monolayer [25] enriched in certain proteins, such as a caveolin-1 mutant form [21]. Jurkat T cells do not express caveolin-1 [1, 2] but reggie-1 and reggie-2 in distinct patterns at the plasma membrane and intracellular compartments.…”

“…7), which is an additional argument for release by exocytosis. The detection of caveolin in the interior of lipid bodies [20,21] and exclusively in the endoplasmic leaflet of ER membranes challenges the classic model of lipid droplet biogenesis. Traditional models assume that lipids initially accumulate between the endoplasmic and cytoplasmic leaflets of the ER membrane and that caveolin molecules reach the surface of the lipid droplet by lateral diffusion in the cytoplasmic leaflet of the ER membrane [20].…”

Raft association and lipid droplet targeting of flotillins are independent of caveolin

“…In particular, it was recently described that caveolins can move from the plasma membrane to lipid bodies in response to oleic acid treatment (Pol et al, 2001(Pol et al, , 2004. In addition, flotillin-1 has been found in purified lipid droplets formed after fatty acid loading in CHO cells (Liu et al, 2004).…”

Regulation of adipose tissue lipolysis revisited

“…Caveolae have several putative functions, including participation in signal transduction (73) , membrane trafficking pathways and NEFA binding and transport (74) . Interestingly, caveolin-1 also associates with LD (75)(76)(77)(78) , hinting at a role for caveolin-1 in lipolysis. Accordingly, caveolin-1-deficient mice display a blunted response to pharmacological and physiological lipolytic stimuli (79) .…”